Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis.

OBJECTIVES: Expression of the nuclear Ki-67 protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore-assisted light inactivation (CALI). MATERIALS AND METHODS: Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target protein. RESULTS: Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicating a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro-injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. CONCLUSIONS: Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.

Ultraviolet light and interleukin-10 modulate expression of cytokines by transformed human dermal microvascular endothelial cells (HMEC-1).

Exposure of the skin to ultraviolet (UV) light causes DNA damage, inflammation, and impairment of local as well as systemic immune responses. Dermal microvascular endothelial cells are key elements for the recruitment of inflammatory cells during the pathogenesis of inflammatory skin diseases via the expression of adhesion molecules and the release of cytokines. Because UVB may directly affect the function of dermal cells it was investigated whether UVB irradiation alters the production of proinflammatory and chemotactic cytokines by endothelial cells. UVB exposure of transformed human microvascular endothelial cells (HMEC-1) resulted in a dose dependently increased mRNA expression as well as release of interleukin (IL)-1beta, IL-6, IL-8, and growth-regulated oncogene alpha (GROalpha). Maximum cytokine production was observed 16-24 h after irradiation when 7.5-12.5 mJ UVB per cm2 were used. In addition, it was examined whether IL-10, which is upregulated in keratinocytes following UVB irradiation and accounts for UV mediated immunosuppression such as inhibition of contact hypersensitivity, also affects endothelial cell cytokine production. Treatment of HMEC-1 with IL-10 significantly enhanced IL-6 and IL-8 release and further upregulated UVB-induced IL-6 and IL-8 mRNA expression. These findings demonstrate that UVB both directly and indirectly via the release of IL-10 stimulates microvascular endothelial cells to produce proinflammatory cytokines and chemokines that are required for the migration and activation of inflammatory cells in UV-mediated inflammatory skin reactions.

Human dermal fibroblasts express prohormone convertases 1 and 2 and produce proopiomelanocortin-derived peptides.

In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopiomelanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. Alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of alpha-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses.

Expression of proopiomelanocortin peptides in human dermal microvascular endothelial cells: evidence for a regulation by ultraviolet light and interleukin-1.

Proopiomelanocortin peptides such as alpha-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte-endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1 beta. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30J per cm(2)) or ultraviolet B (12.5 mJ per cm(2)) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and alpha-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to alpha-melanocyte- stimulating hormone, interleukin-1 beta, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of alpha-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. J Invest Dermatol 115:1021-1028 2000

Alpha-melanocyte stimulating hormone prevents lipopolysaccharide-induced vasculitis by down-regulating endothelial cell adhesion molecule expression.

The neuroendocrine hormone alpha-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that alpha-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFalpha in vitro and in vivo. In HDMEC, alpha-MSH (10(-8)/10(-12) M) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFalpha as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, alpha-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, alpha-MSH effectively inhibited the transcription factor nuclear factor-kappaB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of alpha-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that alpha-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.

Extracellular matrix regulates steady-state mRNA levels of the proliferation associated protein Ki-67 in endometrial cancer cells.

We investigated whether components of the extracellular matrix have the potential to regulate the proliferative activity of endometrial adenocarcinoma cells. Culturing of cells on the reconstituted basement membrane matrigel down-regulated the steady-state mRNA levels of the proliferation associated protein, Ki-67, in the endometrial adenocarcinoma cell lines HEC 1B(L) and Ishikawa after 48-96 h of culture on the matrix substrate. Proliferation of Ishikawa was stimulated again if cells were cultured on matrigel and challenged by proteins representing functional domains of tenascin-C, a mesenchymal glycoprotein. The fibronectin-type-III-like repeats 6-8 of tenascin-C were found to be the most potent. In summary, evidence is provided that components of both epithelial and stromal extracellular matrices can function as regulators of cell growth.

Molecular basis of the alpha-MSH/IL-1 antagonism.

The neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is recognized as a potent mediator of immune and inflammatory reactions. Accordingly, alpha-MSH in vitro, as well as in vivo, antagonizes the proinflammatory activities of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF alpha). Since the molecular basis of these antiinflammatory effects is not well known, the influence of alpha-MSH on IL-1 beta-induced chemokine production and transcription factor activation was investigated in human keratinocytes. alpha-MSH, in a dose-dependent manner, after 48 h, significantly reduced the IL-1 beta mediated secretion of the C-X-C chemokines IL-8 and Gro alpha. This was confirmed by semiquantitative RT-PCR, which revealed a marked down-regulation in IL-8 and Gro alpha mRNA expression. Furthermore, we determined the effect of alpha-MSH on the IL-1 beta-induced activation of the nuclear factor kappa B (NF kappa B)--a major transcription factor for chemokine genes. Electrophoretic mobility-shift-assays showed that alpha-MSH, in a dose range from 10(-6) to 10(-12) M, significantly downregulated the IL-1 beta-induced activation of NF kappa B 10 minutes after stimulation. Therefore, NF kappa B inactivation by alpha-MSH appears to be a crucial event, one that is responsible for the downregulation of cytokine gene transcription.

Mechanisms of the antiinflammatory effects of alpha-MSH. Role of transcription factor NF-kappa B and adhesion molecule expression.

The recruitment of leukocytes from the circulation to inflamed tissue is regulated by the expression of adhesion molecules on both leukocytes and endothelial cells. The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate inflammation. Thus, we investigated the influence of alpha-MSH on the LPS-induced expression of the adhesion molecules E-selectin and VCAM-1 on endothelial cells. Human microvascular endothelial cells (HMEC-1) were treated with LPS (100 ng/ml) alone or in the presence of alpha-MSH (10(-8) to 10(-16) M). RT-PCR analysis showed that alpha-MSH significantly reduced LPS-induced expression of VCAM-1 and E-selectin. Since many adhesion molecules contain regulatory NF-kappa B sites in their promoter region, the role of alpha-MSH in the activation of the transcription factor NF-alpha B was also investigated. alpha-MSH significantly downregulated the LPS-mediated activation of NF-kappa B, in a dose-dependent manner. These findings indicate that modulation of the transcription factor NF-kappa B is a crucial molecular event, one that seems to be responsible for the antiinflammatory effects of alpha-MSH.

Role of epidermal cell-derived alpha-melanocyte stimulating hormone in ultraviolet light mediated local immunosuppression.

Irradiation of the skin with ultraviolet light (UV) results in profound alterations of both local and systemic immune responses. These effects are largely mediated by soluble mediators released from epidermal cells in response to UV. It is well known that keratinocytes release increased amounts of cytokines upon UV-irradiation. UV-light also induces the release of the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha MSH), from keratinocytes, and upregulates the expression of POMC mRNA. alpha MSH exerts a variety of immunomodulating and antiinflammatory effects, mainly by virtue of its capacity to alter the function of antigen presenting cells and vascular endothelial cells. Within an in vivo mouse-model, both intravenous and topical application of alpha MSH resulted in inhibiting the induction, eliciting a contact hypersensitivity reaction, and inducing hapten-specific tolerance. These findings indicate that alpha MSH, released in the epidermis after UV irradiation, may contribute to UV-mediated immunosuppression. The therapeutic application of alpha MSH or alpha MSH-derived peptides may prove to be a useful approach for treating inflammatory skin diseases.

Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone.

The capacity of the skin immune system to mount various types of immune responses is largely dependent on their ability to release and respond to different signals provided by immunoregulatory mediators such as cytokines. There is recent evidence that neuropeptides such as alpha-melanocyte-stimulating hormone (alpha MSH), upon stimulation, are released by epidermal cells including keratinocytes, Langerhans cells, and melanocytes as well as immunocompetent cells. Moreover, alpha MSH recently has been recognized as a potent immunomodulating agent, which inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-1, IL-2, interferon-gamma, downregulates the expression of costimulatory molecules (B7) on antigen-presenting cells; and recently turned out to be a potent inducer of inhibitory mediators such as cytokine synthesis inhibitory factor interleukin-10. Recently, it also was discovered that monocytes among the five known melanocortin (MC) receptors only express MC-1, which is specific for alpha MSH. The expression of MC-1 on monocytes is upregulated by mitogens, endotoxins, and proinflammatory cytokines. There is also recent evidence for the in vivo relevance of the immunosuppressing capacity of alpha MSH. Accordingly, in animals alpha MSH has been shown to inhibit the induction of contact hypersensitivity reactions and to induce hapten-specific tolerance. These findings indicate that, in addition to the cytokine network, neurohormones within the cutaneous microenvironment are a crucial element for the induction, elicitation, and regulation of cutaneous immune and inflammatory responses.

The role of alpha-MSH as a modulator of cutaneous inflammation.

Among various neuropeptides such as substance P, calcitonin gene-related peptide and others, alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC-1R), which is specific for alpha-MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells alpha-MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-2, IFN-gamma, TNF-alpha and IL-1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL-10. On endothelial cells alpha-MSH is capable of downregulating the LPS-induced expression of adhesion molecules such as vascular cell adhesion molecule (VCAM) and E-selectin. Moreover, the LPS-induced activation of transcription factors such as NF kappa B is downregulated by alpha-MSH. In a mouse model i.v. or topical application of alpha-MSH was found to inhibit the induction phase as well as the effector phase of contact hypersensitivity (CHS) reactions and to induce hapten-specific tolerance. These findings indicate that the production of immunosuppressing neuropeptides such as alpha-MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

Expression of functional melanocortin receptors and proopiomelanocortin peptides by human dermal microvascular endothelial cells.

Human dermal microvascular endothelial cells (HDMEC) are capable of mediating leukocyte-endothelial interactions by the expression of cellular adhesion molecules and the release of proinflammatory cytokines and chemokines during cutaneous inflammation. Recent studies support the important role for proopiomelanocortin (POMC) peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), as immunomodulators in the cutaneous immune system. The purpose of the studies described here was to determine whether HDMEC serves as both target and source for POMC peptides. RT-PCR and Northern blot studies demonstrated the constitutive expression of mRNA for the adrenocorticotropin (ACTH) and alpha-MSH-specific melanocortin receptor 1 (MC-1R) in HDMEC, and the microvascular endothelial cell line HMEC-1 that could be upregulated by stimulation with IL-1 beta and alpha-MSH. HDMEC responded to stimulation by alpha-MSH with a dose- and time-dependent synthesis and release of the CXC chemokines, IL-8 and GRO alpha. Likewise, alpha-MSH augmented HDMEC chemokine release induced by TNF or IL-1. HD-MEC were found to constitutively express POMC and prohormone convertase 1 (PC-1); the latter being required to generate ACTH from the POMC prohormone. POMC and PC-1 mRNA expression are increased as a result of stimulation with UVB and UVA1 radiation, IL-1, and alpha-MSH. In addition, UV-radiation is capable of inducing the release of HDMEC, ACTH, and alpha-MSH in a time- and dose-dependent fashion. Thus, these data provide evidence that HDMEC are capable of expressing functional MC-1R, POMC, and PC-1 mRNA; and of releasing POMC peptides with UV light, IL-1, and alpha-MSH as regulatory factors. The expression and regulation of these peptides may be of importance, not only for the autocrine or paracrine regulation of physiologic functions of dermal endothelial cells, but also for the regulation of certain microvascular-mediated cutaneous or systemic inflammatory responses.

Molecular characterization of a Theileria lestoquardi gene encoding for immunogenic protein splice variants.

A Theileria lestoquardi schizont cDNA library was screened using sera collected from sheep recovering from a natural malignant theileriosis infection. An immunogenic clone (clone-5) was isolated and its full sequence was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR) technique. PCR experiments and sequencing demonstrated the presence of two transcript forms of the gene, resulting from splicing variation at the single intron found in the gene. Both gene products, clone-5 long and clone-5 short variants with calculated molecular weights of 99.9 and 72.7 kDa, respectively, were expressed in a T. lestoquardi-infected cell line. BLAST searches suggested the presence of homologues of the gene in both the Theileria parva and Theileria annulata genomes, with identities of 53 and 62% on the DNA level, respectively. The intron was preserved in size, sequence, and location within the gene in these parasites. Analysis of the subcellular localization of the clone-5 proteins showed a predominant parasite membrane association in T. lestoquardi-infected cells. Both recombinantly produced forms were found to be reactive with sera from infected animals. Bioinformatic analyses were employed to address the possible function of the gene products in the biology of T. lestoquardi.

Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui.

The nucleotide sequence has been determined of a 4700 bp region from a ribosomal protein gene cluster of Halobacterium marismortui (Haloarcula marismortui), which is equivalent to part of the spectinomycin operon of Escherichia coli. The genes were localized on the recombinant lambda EMBL3 clone PP*7, which also contains several other ribosomal protein genes from the DNA region in H. marismortui equivalent to the linked S10/spc operon. The genes analysed encode ten ribosomal proteins, namely HmaL5, HmaS14, HmaS8, HmaL6, HL5, HL24, HmaL18, HmaS5, HmaL30 and HmaL15. The gene organization of the archaebacterial cluster is similar to that in eubacteria but has two additional genes, namely those encoding HL5 and HL24, which were identified as extra proteins that are apparently not present in E. coli. These correspond to the gene products of orfd and orfe in Methanococcus vannielii and also have eukaryotic counterparts.

The alpha-operon equivalent genome region in the extreme halophilic archaebacterium Haloarcula (Halobacterium) marismortui.

The genome region of the extreme halophilic archaebacterium Haloarcula marismortui equivalent to the alpha-operon of Escherichia coli has been characterized. In H. marismortui, the alpha-operon was found to be located immediately upstream from the S9 gene cluster. The gene order in the halobacterial alpha-operon, given according to the gene products, is tRNA(Ser), HmaS13, HmaS4, HmaS11, and HmaRp alpha. Compared to the corresponding operon from E. coli, the halobacterial gene organization differs in (i) the presence of a gene for tRNA(Ser) (GCU), (ii) the reversed order of the genes for the ribosomal proteins HmaS11 and HmaS4, and (iii) the absence of the gene coding for the ribosomal protein L17. The primary structure of HmaRp alpha shows high similarity to a subunit of eukaryotic RNA polymerase II (YeaRpB3, HsaRpB33), whereas the similarity to the eubacterial alpha-subunit of RNA polymerase is only weak.

The Ki-67 protein: from the known and the unknown.

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.

The role of alpha-melanocyte-stimulating hormone in cutaneous biology.

alpha-Melanocyte stimulating hormone is a neuroimmunomodulating peptide that was recently detected in many non-pituitary tissues including the skin. Accordingly, epidermal cells such as keratinocytes and melanocytes (as well as dermal cells such as fibroblasts and endothelial cells), after stimulation with pro-inflammatory cytokines or UV light, synthesize, and release alpha MSH. The effects of these peptides are mediated through specific melanocortin (MC) receptors that can be detected on immunocompetent and inflammatory cells as well as on keratinocytes, melanocytes, fibroblasts, and endothelial cells. In addition to its well known pigment-inducing capacity, alpha MSH is able to modulate keratinocyte proliferation and differentiation. Endothelial cell and fibroblast cytokine production and fibroblast collagenase production are also regulated by alpha MSH. The immunosuppressive capacity of alpha MSH is mediated mainly through its effects on monocyte and macrophage functions. Accordingly, alpha MSH downregulates the production of pro-inflammatory cytokines and accessory molecules on antigen-presenting cells. The production of suppressor factors such as IL-10, however, is upregulated by alpha MSH. The in vivo relevance of these data is documented by the finding that systemic application of alpha MSH inhibits the induction and the elicitation of murine contact hyper-sensitivity and induces hapten-specific tolerance. These findings indicate that alpha MSH is part of the mediator network that regulates cutaneous inflammation and hyper-proliferative skin diseases.

Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone.

Pro-opiomelanocortin (POMC)-derived peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) recently have been recognized as mediators with potent immunomodulating and anti-inflammatory properties. Their effects are mediated via different protein G-coupled melanocortin (MC) receptors that are capable to bind one or more POMC-derived peptides. Among these receptors, MC-1 is specific for alpha-MSH and adrenocorticotropin. The purpose of the present study was to investigate whether MC receptors are expressed on normal human dermal microvascular endothelial cells (HDMEC) as well as transformed human dermal microvascular endothelial cells (HMEC-1). Using semiquantitative reverse transcriptase-PCR and MC receptor-specific primers, both HDMEC and HMEC-1 were found to express MC-1 constitutively. In addition, MC-1 expression was increased upon stimulation with IL-1beta or alpha-MSH itself. Other known MC receptors were neither detectable in unstimulated nor in IL-1beta- or alpha-MSH-stimulated cells. The binding of alpha-MSH by HMEC-1 was specific and saturable as demonstrated by competitive and saturation-binding studies with 125I-labeled alpha-MSH (Kd: 1.1 nM). To evaluate the physiologic relevance of MC-1 expression, HMEC-1 were treated with various concentrations of alpha-MSH (10(-15)-10(-6) M) and were investigated for their cytokine-producing capacity. Alpha-MSH (10(-10)-10(-8) M) significantly up-regulated IL-8 release and mRNA expression by HMEC-1. In contrast, the production of IL-1 or IL-6 by HMEC-1 was not affected upon treatment with alpha-MSH. These data provide first evidence that HDMEC express functional MC receptors. Therefore, alpha-MSH, which is released in the skin during cutaneous inflammation via inducing chemokines may represent an important signal required for leukocyte-endothelial cell interaction.